SDS-PAGE and Immunoblotting of Liver Microsomes

Liver microsomal samples were adjusted to 1 mg/ml in LDS Lithium Dodecyl Sulfate sample buffer (Life Technologies) and subjected to SDS-PAGE and immunoblotting as previously described (MacLeod et al., 2015 (link)). Primary antibodies were used as follows: anti-CYP1A1 (1:1000; AB1258; Millipore, Watford, United Kingdom) and Cyp1a (1:1000) (Forrester et al., 1992 (link)). Anti-GRP78 (1:1000; ab21685; Abcam (Cambridge, UK)) was used as the loading control. Immunoreactive proteins were detected using polyclonal goat anti-rabbit or anti-mouse horseradish peroxidase immunoglobulins as secondary antibodies (Dako, Ely, United Kingdom) and were visualized using Immobilon chemiluminescent substrate (Millipore) and a Fujifilm LAS-3000 mini-imaging system (Fujifilm UK Ltd., Bedford, United Kingdom).

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Lin D., Kostov R., Huang J.T., Henderson C.J, & Wolf C.R. (2017). Novel Pathways of Ponatinib Disposition Catalyzed By CYP1A1 Involving Generation of Potentially Toxic Metabolites. The Journal of Pharmacology and Experimental Therapeutics, 363(1), 12-19.

Publication 2017

LDS Lithium Dodecyl Sulfate sample buffer AB1258 ab21685 Immobilon chemiluminescent substrate Fujifilm LAS-3000 mini-imaging system

Anti antibodies Anti immunoglobulins Antibodies Buffer Cyp1a1 Goat Grp78 Horseradish peroxidase Immobilon Immunoglobulins Lithium dodecyl sulfate Liver microsomal Mouse Proteins Rabbit Sds page

Corresponding Organization :

Other organizations : University of Dundee, Ninewells Hospital

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Variable analysis

independent variables

Liver microsomal samples

dependent variables

Expression levels of CYP1A1 protein

control variables

Protein concentration of liver microsomal samples (1 mg/ml)
Use of LDS Lithium Dodecyl Sulfate sample buffer
SDS-PAGE and immunoblotting protocol
Primary antibodies: anti-CYP1A1 (1:1000), Cyp1a (1:1000)
Loading control: anti-GRP78 (1:1000)
Secondary antibodies: polyclonal goat anti-rabbit or anti-mouse horseradish peroxidase immunoglobulins
Visualization using Immobilon chemiluminescent substrate and Fujifilm LAS-3000 mini-imaging system

positive control

Not explicitly mentioned

negative control

Not explicitly mentioned

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